In this blog post, I begin to describe the HSV-Type-Specific ABVIC Test. Regarding the acronym ABVIC, this stands for “Antibody-Binding to Virus-Infected Cells.” For starters, I address four key questions about the ABVIC Test which has been under development since 2011 and which is now patented and soon-to-be licensed by my company, Rational Vaccines (RVx), Inc.
- How does ABVIC compare to the Current Herpes Antibody Tests?
- Practical Advantage of HSV-Type-Specific ABVIC?
- Why is the HSV-Type-Specific ABVIC Test Better?
- What do HSV-Type-Specific Test Results Look Like?
1. how does ABVIC compare to CURRENT HerpeS ANTIBODY TESTs?
My laboratory is currently in the process of finalizing and writing up a manuscript for publication that will compare the resolving power of the HSV-Type-Specific ABVIC Test to the available Herpes Antibody Tests, HerpeSelect and HSV Western Blot. I attach a 1-page poster of the results of this study here: Autumn Immunology Conference Poster (Nov 2015) This poster was presented at the Autumn Immunology Conference in Chicago in Nov 2015 and will be presented at the meeting of the American Association of Immunologists in Seattle in May 2016.
In this blog post, I summarize the results of the ABVIC analysis prior to its formal publication, because I think this news is too important for the potentially millions of people who have been told by a doctor or nurse over the past 20 years that they “might have HSV-2,” based on what are scientifically flimsy test results (i.e., HerpeSelect Index Values between 1 and 5). Within the next 3 to 4 months (pending CLIA certificiation), Rational Vaccines (RVx), Inc will be offering the HSV-Type-Specific ABVIC Test to patients whom have received past herpes antibody results that suggest they are infected with HSV-2 despite an absence of clinical signs or behavioral risk factors, then you may wish to consider RVx’s HSV-Type-Specific ABVIC Test (cost $250). RVx’s website will be live by Summer 2016. To sign up for a better HSV antibody test today, please send an email stating your name, phone number, and a permanent email address to “firstname.lastname@example.org,” and RVx will contact you once the CLIA certification of our new diagnostic facility is complete.
Below, I explain why the HSV-Type-Specific ABVIC Test is head-and-shoulders better than any other HSV Antibody test currently on the market.
2. PRACTICAL Advantage of HSV-Type-specific abvic?
The results I communicate in this section are not theoretical or based on animal studies. Rather, these are studies performed with actual human patients. As described to and approved by my university’s institutional review board, my lab has received 45 serum samples over the past year from Westover Heights Clinic in Portland, Oregon in collaboration with Terri Warren.
17 of the serum samples were controls: ♦ HSV seronegative; ♦ HSV-1-seropositive; ♦ HSV-2 seropositive; or ♦ HSV-1,2-double-seropositive.
The other 28 serum samples were from individuals who had received equivocal results that implied, but did not prove, they were life-long carriers of the HSV-2 virus. Specifically, each of these patients had been told they might have HSV-2 because they received an Indeterminate diagnosis from antibody tests (often multiple rounds of tests). Specifically, most patients would first receive the following results:
- A HerpeSelect test that indicated they were low-HSV-2 antibody positive (Index Value of 1 to 5); and everyone in our study would have secondarily ordered a………
- Herpes Western Blot which had yielded Indeterminate results because “ghost bands” appeared at unexpected positions on the blot calling into question if the bands in the HSV-2 lane really represented the presence of HSV-2-specific antibodies or were just more “ghost bands” [i.e., high background).
In the first 34 serum samples sent by the Westover Heights Clinic, my lab had no prior knowledge of which samples were which, and we performed a blinded analysis with our new HSV Type-Specific ABVIC Test. All of the control serum samples were correctly and immediately diagnosed by the ABVIC Test in 100% agreement with HerpeSelect and HSV Western Blot (i.e., these are the easy samples that all of the tests get right). In contrast, 16 of the 17 Indeterminate Samples that had confounded both HerpeSelect and HSV Western Blot Tests proved to be HSV-2 seronegative. Each of these serum samples represents one person who was informed by the Current Standard of Herpes Antibody Testing led that they “might” have HSV-2 genital herpes, and they should live their life accordingly and not risk transmitting the virus (that they did not carry) to others. However, in these 16 of 17 cases, the quantitatively superior HSV-Type-Specific ABVIC revealed the plain and simple truth that these patients simply had serum that yielded “High Background” on all three of the antibody tests (HerpeSelect, Western, and ABVIC). Because the ABVIC test is internally controlled, high background is easily distinguished from “low HSV-2-positive.” To date, of 25 Indeterminate samples screened, only 1 of 25 is actually low HSV-2-positive which means that the vast majority of HerpeSelect tests that yield Index Values in the range of 1 to 4 are most likely false-positives. With the creation of the new HSV-Type-Specific ABVIC test, there is now a simple test to resolve likely HSV-2-false-positive diagnoses.
3. Why is HSV-Type-Specific ABVIC Better?
HerpeSelect and HSV Western blot tests are what is known as qualitative tests, or +/- tests. Qualitative tests are fine provided that what you are testing for is black or white with no gray are in between. However, in practice, when one makes scientific measurements (such as measuring the amount of HSV-2-specific antibody in blood), there will be many samples that fall in the gray area between the extremes. It is here in this gray area that qualitative (+/-) tests are prone to fail because it becomes a subjective guess of the observer (scientist) as to where the dividing line between “negative” and “positive” should be drawn.
In contrast to HerpeSelect and HSV Western Blot, the HSV-Type-Specific ABVIC Test is highly quantitative over a 100-fold dynamic range of measurements and it is the highly quantitative nature of the test that makes it far more robust, objective, and amenable to statistical analysis such that the threshold between “negative” and “positive” may be more reliably drawn. So what scientific facts back up this claim that the HSV-Type-Specific ABVIC Test is a more quantitatively reliable test?
- Only the ABVIC Test is internally controlled, which allows the test to differentiate “high background” patient serum samples from patient serum that contains “low levels of HSV-2-specific antibodies.” In the absence of an internal control, it is not surprising that 5% of HerpeSelect tests will mislead patients and/or doctors into believing they have HSV-2 because “high background serum” will return a low-positive index value in the range of 1 to 5.
- Only the ABVIC Test uses a flow cytometer that can sample 1000s of test cells per second. Thus, each ABVIC Test can compare the efficiency with which a patient’s antibodies bind to (1) uninfected background control cells versus (2) HSV-1+ cells versus (3) HSV-2+ cells. The preferential binding of a patient’s serum antibodies to thousands of HSV-2+ cells may be compared to thousands of uninfected background controls in 20 seconds such that each “DIAGNOSIS” is based upon tens of thousands of quantitative measurements, which are then subjected to statistical analysis so that a patient may learn the probability that they are infected with HSV-2. In carpentry, the idiom goes “Measure twice, cut once.” In arriving at the correct conclusion of whether or not a person possesses blood antibodies to HSV-2, I would suggest that procedurally it makes the most sense to make 1000s of quantitative measurements of the level of patient antibody that binds all 75 proteins in HSV-2-infected cells (full antigenic breadth) versus antibody binding to uninfected cells that serve as an internal background control. This is what the HSV Type-Specific Test offers, and our forthcoming publication on a study of human subjects suggests that the method can reduce the rate of false-positive HSV-2 diagnoses by at least 90%. RVx will be offering the HSV-Type-Specific ABVIC Test for patients in need of this service at a cost of $250, and the test can be completed discreetly in less than two weeks.
RVx’s HSV-Type-Specific ABVIC Test (cost $250). RVx’s website will be live by Summer 2015. To sign up for a better HSV antibody test today, please send an email stating your name, phone number, and a permanent email address to “email@example.com,” and RVx will contact you once the CLIA certification of our new diagnostic facility is complete.
4. WHAT DO HSV-Type-Specific Test RESULTS LOOK LIKE?
In a subsequent post, I will explain in detail how the HSV-Type-Specific ABVIC Test works. For now, I simply describe the type of results a patient would receive and I will try to keep the technical details to a minimum.
The basis of the test are fixed and permeabilized cells (i.e., stable cells with lots of holes in them so antibodies can access nearly all of the proteins inside the cells). All of the descriptive words that follow refer to the Figure below of a representative patient’s results. In total, three different ABVIC tests are run, and these are (1) the ABVIC^HSV test which tests for total antibodies to HSV-1 and/or HSV-2; (2) the ABVIC^HSV-1 test which tests for only HSV-1-specific antibodies; and (3) the ABVIC^HSV-2 test which tests only for HSV-2 -specific antibodies.
Three populations of cells are included in all of the test and these are:
- Uninfected cells that contain no viral proteins. In the graphs below, these are shown on the left and are labeled “UI cells.“
- HSV-1-infected cells that contain all of HSV-1’s ~75 proteins. In the graphs below, these are shown in the middle and are labeled “HSV-1+ cells“
- HSV-2-infected cells that contain all of HSV-2’s ~75 proteins. In the graphs below, these are shown on the right and are labeled “HSV-2+ cells“
The flow cytometer can distinguish these three populations of cells because of a trick we use in the lab called CFSE-differential labeling. It does not matter how it is done, but it allows the flow cytometer to segregate the three populations into three separate columns.
Next, a small dilution of the patient’s serum (cell-free fraction of blood) is added to the “Test Cells (UI cells + HSV-1 cells + HSV-2 cells) and the antibodies are allowed to bind any of the 75 viral proteins present in HSV-1+ or HSV-2+ cells. In addition, at some rate antibodies will non-specifically stick to all of the cells and this is called the “background” of the test. The background of the test for a given sample is defined by the amount of antibody that sticks to the “UI cells” which serve as an internal background control.
Excess patient antibodies are rinsed from test cells and a “far-red secondary antibody” that binds human IgG antibody (potentially stuck to HSV-2 proteins) is added to the test cells. The more HSV-1 or HSV-2-specific antibody present in a patient’s serum sample, the more human antibodies are stuck to the HSV-1+ cells and/or HSV-2+ cells and the more of the “far-red secondary antibody” gets stuck to those same cells. At the end of this second labeling step, excess secondary “far-red” antibody is washed from the test cells.
KEY CONCEPT DEFINED HERE: The more “HSV-specific antibody a person has in their serum,” the more far-red color will preferentially stick to the HSV-1+ or HSV-2+ cells compared to UI cells, and thus the higher those cell populations will “shift up” the y-axis……..higher on the y-axis equals brighter red = more serum antibody stuck to a sub-population of test cells.
TEST RESULTS EXPLAINED FOR JOHN A. DOE
In the first ABVIC^HSV Test at the top of the Figure, serum from John A. Doe was combined with the Test cells and revealed that this person is “HSV-seropositive” because both HSV-1+ cells and HSV-2+ cells are shifting upwards. This first test does not distinguish whether this person has HSV-1 or HSV-2, but the test results indicate that the probability of this person being HSV-seronegative (not infected with either virus) is less than one in a million.
In the second ABVIC^HSV-1 Test in the middle of the Figure, serum from John A. Doe was first pre-absorbed to HSV-2+ cells to remove all (1) type-common antibodies shown as blue ‘Y’s in the top right box and to remove and (2) HSV-2-specific antibodies shown as green ‘Y’s in the top right box. Hence, the only population of HSV-specific antibodies that could be left would be the red ‘Y’s that are HSV-1-specific antibodies. In the case of John A. Doe, the individual has no HSV-1-specific antibodies as shown in the middle graph because the HSV-1+ test cells are no “redder’ (shifted up the y-axis) than the HSV-2+ test cells. Based on statistical analysis, John A. Doe is HSV-1-seronegative.
In the third ABVIC^HSV-2 Test at the bottom of the Figure, serum from John A. Doe was first pre-absorbed to HSV-1+ cells to remove all (1) type-common antibodies shown as blue ‘Y’s in the top right box and to remove and (2) HSV-1-specific antibodies shown as red ‘Y’s in the top right box. Hence, the only population of HSV-specific antibodies that could be left would be the green ‘Y’s that are HSV-2-specific antibodies. In the case of John A. Doe, the individual has HSV-2-specific antibodies as shown in the bottom graph because the HSV-2+ test cells are “redder’ (shifted up the y-axis) than the HSV-1+ test cells. Based on statistical analysis, John A. Doe has less than a one-in-a-million probability of being HSV-2-seronegative. Hence, John Doe’s blood test offers definitive proof that he is infected with the HSV-2 virus.
In subsequent posts, I will consider the HSV-Type-Specific ABVIC Test in greater depth and examine the underlying reason that it is 100 times more sensitive than the current standard of care in herpes antibody testing around the world; namely, Focus Diagnostics’ HerpeSelect Test.
– Bill H.